Facile peptide thioester synthesis via solution-phase tosylamide preparation

Article abstract of DOI:10.1016/j.tetlet.2006.11.143

Preparation of peptide thioester is essential for native chemical ligation and block condensation. Our novel methodology involves conversion of the carboxylic acid of a peptide into a thioester using p-toluenesulfonyl isocyanate, followed by alkylation, t

Full text of DOI:10.1016/j.tetlet.2006.11.143

Tetrahedron Letters 48 (2007) 849–853
Facile peptide thioester synthesis via solution-phase
tosylamide preparation
b
a,
*
Shino Manabe,a,b, Tomoyuki Sugioka and Yukishige Ito
*
aRIKEN (The Institute of Physical and Chemical Research), Hirosawa, Wako, Saitama 351-0198, Japan
bPRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-1102, Japan
Received 9 October 2006; revised 20 November 2006; accepted 24 November 2006
Available online 13 December 2006
Abstract—Preparation of peptide thioester is essential for native chemical ligation and block condensation. Our novel methodology
involves conversion of the carboxylic acid of a peptide into a thioester using p-toluenesulfonyl isocyanate, followed by alkylation,
then thiol substitution. Our methodology can also be used for the preparation of glycopeptide thioesters. Furthermore, it is possible
to carry out the reaction as a sequential peptide chemical ligation.
Ó 2006 Elsevier Ltd. All rights reserved.
Recently, peptide thioesters have gained attention as
intermediates in peptide synthesis because of their utility
towards thioester peptide block coupling and native
chemical ligation methods.1 Although peptide thioesters
are essential for native chemical ligation or block cou-
pling methodology, the chemical synthesis of C-terminal
thioester peptides via traditional Fmoc-based solid-
phase strategy has been hampered by the poor stability
of the thioester bond under basic conditions, which
are required for the deprotection of the N-Fmoc group.
It is highly desirable to develop a synthetic scheme that
is compatible with Fmoc solid-phase peptide synthesis.
ditions and nucleophiles.4 In the case of the peptide thio-
ester formation using Kenner’s linker, as reported by
Pessi and co-workers, upon completion of the elonga-
tion process, the peptide was cleaved by a thiolate attack
as the corresponding thioester.5 Subsequently, Bertozzi
and co-workers applied this methodology for the syn-
thesis of a glycopeptide.6 Although the safety-catch type
linker methodology is conceptually elegant, difficulties
have been recently reported. For example, the attach-
ment of the first amino acid to the sulfonamide Kenner’s
linker resulted in poor coupling yields and racemiza-
tion.7 In regards to glycopeptide synthesis, Unverzagt
reported on the limitations of the Kenner’s sulfonamide
linker during the capping step,8 and Bertozzi and
co-workers reported on the unsuccessful glycopeptide
synthesis using a combination of the safety-catch linker
and solid-phase Fmoc peptide synthesis.9 As a note,
Camarero has recently introduced an aryl hydrazine lin-
ker as a safety-catch linker.10 The third approach for
peptide thioester preparation involved the creation of
the thioester group after cleaving the peptide from resin.
This approach appears to be most practical because con-
ventional peptide synthetic methodology is fully uti-
lized.11 However, there are some reports involving this
approach, for which a satisfactory methodology is
needed. Sewing and Hilvert has reported on the use of
Me3Al–thiophenol as a releasing reagent from the resin;
unfortunately, concomitant formation of aspartimide at
Recent examples of peptide thioester preparations can
be classified into three synthetic approaches: the first
scheme attempts to attenuate the basicity during cleav-
age of the Fmoc group with direct attachment of the
thioester to the solid-phase resin. For example, Aimoto
synthesized a 25-mer thioester peptide using a combina-
tion of 1-methylpyrrolidine–hexamethyleneimine–HOBt
as the Fmoc cleavage reagent to prevent cleavage of the
thioester from the resin.2 Unfortunately, the thioester
racemized more readily than other esters under basic
conditions.3 The second approach for peptide thioester
preparation is based on Kenner’s safety-catch linker,
which is particular by stable under basic and acidic con-
the aspargine residue proved to be problematic.12
A
Keywords: Thioester; Glycopeptide; Sequential peptide ligation.
*
novel method for the preparation of a thioester using
Corresponding author. Tel.: +81 48 467 9432; fax: +81 48 462
4680; e-mail: smanabe@riken.jp
a trithioorthoester was reported by Brask et al.13
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.11.143

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S. Manabe et al. / Tetrahedron Letters 48 (2007) 849–853
However, its utility is restricted mainly to the prepara-
tion of glycine thioesters. Botti and Danishefsky have
reported the peptide thioester formation by O–S acyl
shift and its application to the synthesis of a large pep-
tide.14 As well as O–S shift, N–S acyl shift strategy
was also reported recently.15 We report here the facile
preparation of sulfonamides from carboxylic acids using
p-toluenesulfonyl isocyanate.
preparation of tosylamides, conversion to the corre-
sponding thioester was carried out according to a
reported procedure based on Kenner’s linker on solid-
phase resin. When tosylamide 2a was treated with
TMSCHN2, N-methylated 3a and O-methylated 6 were
obtained in 63% and 18% yields, because of the nature
of delocalized N-acylsulfonamide.16 In constant, methyl-
ation using MeI in the presence of K2CO3 resulted in the
desired N-methylated 3a in a high yield. Similarly, alkyl-
ation using iodoacetonitrile was also successful giving
N-alkylated 4a in a good yield, but requiring a longer
reaction time. It was found that microwave irradiation
enhanced the reaction rate dramatically. The alkylation
reaction was completed within 10 min under microwave
N-Protected amino acids 1a–c were converted to the
corresponding tosylamide in quantitative yields (Table
1). Typical N-protecting groups such as Boc, Fmoc
and Cbz have been shown to be stable under these con-
ditions. Subsequent to the establishment of the facile
Table 1. The preparation of amino acid thioesters
R
R
R
R
SBn
PGHN
CO2H
PGHN
CONHTs
PGHN
CONTs
R'
PGHN
O
1
2
3 or 4
5
Entry
Acid
Tosylamide yield (%)
Alkyl tosylamide yield (%)
Thioester yield (%)
SBn
CbzHN
CbzHN
CO2H
CbzHN
CONTs
Me
CbzHN
CONHTs
O
5a
1a
3a
2a
quant.
90 (from 3a)
71 (from 4a)
63 (method A)
83 (method B)
1
CbzHN
CONTs
4a
CH2CN
70 (method C)
76 (method D)
Ph
Ph
FmocHN
1b
Ph
FmocHN
Ph
SBn
FmocHN
3b
CONTs
Me
CO2H
CONHTs
FmocHN
5b
2
O
2b
68 (method A)
95 (method E)
quant.
89 (from 3b)
O
CO2H
CONTs
CONHTs
SBn
N
N
N
N
Me
Boc
Boc
5c
Boc
Boc
1c
3c
2c
90 (from 3c)
82 (from 4c)
84 (method B)
quant.
3
CONTs
N
CH2CN
Boc
4c
90 (method C)
NTs
OMe
CbzHN
6
Method A: TMSCHN2, MeOH, PhH, room temperature, Method B: MeI, K2CO3, DMF, 60 °C, overnight, Method C: ICH2CN, K2CO3, DMF,
60 °C, overnight, Method D: ICH2CN, K2CO3, DMF, microwave irradiation, 300 W, 1 bar, <10 min, Method E: trimethyloxonium tetrafluoro-
borate, i-Pr2NEt, DMF.

S. Manabe et al. / Tetrahedron Letters 48 (2007) 849–853
851
AcO
AcO
AcO
AcO
O
O
AcO
O
O
AcO
CO2Bn
COSBn
O
O
NHAc
NHAc
H
H
N
N
HN
N
HN
N
i), ii)
O
O
O
OBn
O
OBn
N
N
O
O
BnO
BnO
NHBoc
NHBoc
8
7
Scheme 1. The synthesis of glycopeptide thioester. Reagents and conditions: (i) H2, Pd/C (en), MeOH, then p-toluenesulfonyl isocyanate, Et3N,
THF, 87%; (ii) TMSCHN2, MeOH, PhH, then benzyl mercaptane, i-Pr2NEt, DMF, 57% (two steps).
irradiation. Compound 3a reacted with benzyl mercap-
tan in the presence of i-Pr2NEt in DMF to give thioester
5a in a high yield very smoothly. Similarly, the reaction
of Fmoc-protected amino acid 3b was also successful.
The yield of 3b was increased by a combination of tri-
methyloxonium tetrafluoroborate–iPr2NEt at room
temperature for 5 min to 95%. Comparatively, cyano-
methylated compounds 4 were more reactive to nucleo-
philic attacks than methylated compounds 3. Thus, the
reaction of 4a and 1.5 equiv of benzyl mercaptan was
completed within 20 min and afforded the correspond-
ing thioester in 71% yield, while in the case of 3a, the
reaction was not completed within 20 min and afforded
the corresponding thioester in 90% yield. The O-alkyl-
ated product 6 was not a substrate for substitution reac-
tion of benzyl mercaptan and was recovered unchanged
quantitatively. Based on the above results, it appears
that the safety-catch linker may be attributable to the
alkylation step—in other words, N-selective alkylation
is required.17
tide synthesis.18 After selective removal of benzyl ester
of 7 by Pd/C(en) under hydrogen atmosphere,19 carb-
oxylic acid was converted to sulfonamide in 87% yield
(Scheme 1). After subsequent activation of sulfonamide
by TMSCHN2 in MeOH–PhH, the corresponding benz-
yl thioester 8 was synthesized in 57% yield in two steps.
The glycopeptide sequence is a part of repeating unit at
the C-terminus of RNA polymerase II, and O-GlcNAc
is suggested to play an important role in transcription
process.20
Next, to demonstrate the advantage of our methodol-
ogy, various substituents were introduced into the thiol
moiety (Scheme 2). Besides benzyl mercaptan, an ester
group with a long alkyl thiol 9 and HSCH2PPh2 1021
gave the corresponding thioesters in high yields under
similar conditions. Thiol unprotected cysteine 11
afforded a dipeptide in 85% yield in 5 min; however, it
remains unclear whether the amino group reacted
directly or thioester 15 was an intermediate.
Because the utility of glycopeptide thioester is obvious,
we then set out to apply our methodology to glycopep-
Since thioester derivatives are now readily available,
sequential ligation approach can be achieved in a
S
PPh2
CbzHN
O
13
b)
H
a)
c)
CO2Me
S
N
CO2Et
SH
CbzHN
CbzHN
CbzHN
CONTs
O
O
14
CH2CN
12
4a
HS
HS
CO2Me
HS
PPh2
H2N
CO2Et
9
10
11
NH2
CO2Et
S
14
CbzHN
O
15
Scheme 2. Reagents and conditions: (a) 9, i-Pr2NEt, DMF, 70%; (b) 10, i-Pr2NEt, DMF, 87%; (c) 11, i-Pr2NEt, DMF, 81%.

852
S. Manabe et al. / Tetrahedron Letters 48 (2007) 849–853
Boc Asp(OMe) Gly OH
16
Boc Gly Thr(Bn) Gly OH
17
i)-iii)
iv)-vi)
Boc Asp(OMe) Gly S
NO2
+
H Gly
Thr(Bn) Gly SBn
18
19
vii)
+
Boc Asp(OMe) Gly Gly Thr(Bn) Gly SBn
H
Thr(Bn) Lys(Cbz) OMe
21
20
vii)
Boc Asp(OMe) Gly Gly Thr(Bn) Gly Thr(Bn) Lys(Cbz) OMe
22
Scheme 3. Reagents and conditions: (i) H2, 10% Pd/C, MeOH; then p-toluenesulfonyl isocyanate, Et3N, THF, 91% (two steps); (ii) ICH2CN,
K2CO3, DMF, 75%; (iii) p-nitrothiophenol, i-Pr2NEt, DMF, quant.; (iv) H2, 10% Pd/C, MeOH, then p-toluenesulfonyl isocyanate, Et3N, THF 68%
(two steps); (v) MeI, K2CO3, DMF, 92%; (vi) BnSH, i-Pr2NEt, DMF, quant.; (vii) i-Pr2NEt, DMF, 64%; (viii) 20, 21, HOOBt, AgCl, i-Pr2NEt,
DMF, 64%.
chemoselective manner (Scheme 3).22 The first peptide
segment 18 has a highly selective p-nitrophenyl thioester,
Seebach, D. J. Peptide Res. 2005, 65, 229–260; (d) Nilsson,
B. L.; Soellner, M. B.; Raines, R. T. Annu. Rev. Biophys.
Biomol. Struct. 2005, 34, 91–118; (e) Muralidharan, V.;
whereas the middle segment 19 was benzyl thioester
Muir, T. W. Nat. Methods 2006, 3, 429–438.
moiety. Both thioesters 18 and 19 were prepared from
carboxylic acids 16 and 17 using our methodology in
high yields. The first ligation between p-nitrophenyl thio-
ester 18 and amino group carrying benzyl thioester 19
2. (a) Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett.
1998, 39, 8669–8672; (b) Clippingdale, A. B.; Barrow, C.
J.; Wade, J. D. J. Pept. Sci. 2000, 6, 225–234; (c) Bu, X.;
Xie, G.; Law, C. W.; Guo, Z. Tetrahedron Lett. 2002, 43,
was carried out in DMF, which was complete within
30 min at room temperature.23 The final product 22,
which is a part of a glycosaminoglycan-binding protein
Midkine,24 was obtained in 64% yield by the subsequent
reaction between amine 21 and terminal thioester 20
by AgCl–HOOBt (3,4-dihydro-3-hydroxy-4-oxo-1,2,3-
benzotriazine) activation.25–27
2419–2422.
3. Hasegawa, K.; Sha, Y. L.; Bang, J. K.; Kawakami, T.;
Akaji, K.; Aimoto, S. Lett. Pept. Sci. 2002, 8, 277–284.
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In conclusion, the novel methodology for peptide thio-
ester preparation was demonstrated. The sequential
peptide ligation was also demonstrated. Our peptide
thioester synthesis can readily accommodate to the stan-
dard Fmoc as well as Boc-based peptide synthetic strat-
egy. Our methodology is operationally simple, and we
believe that it will find practical use and be highly uti-
lized for preparing thioesters of various chemical groups
such as glycoside, phosphates, biotins and fluorescent
chromophores.
8. Mezzato, S.; Schaffrath, M.; Unverzagt, C. Angew. Chem.,
Int. Ed. 2005, 44, 1650–1654.
9. Marcaurelle, L. A.; Mizoue, L. S.; Wilken, J.; Oldham, L.;
Kent, S. B. H.; Handel, T. M.; Bertozzi, C. R. Chem. Eur.
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A. R. J. Org. Chem. 2004, 69, 4145–4151.
11. von Eggelkraft-Gottanka, R.; Klose, A.; Beck-Sickinger,
A. G.; Beyermann, M. Tetrahedron Lett. 2003, 44, 3551–
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3395–3396.
13. Brask, J.; Albericio, F.; Jensen, K. J. Org. Lett. 2003, 5,
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14. (a) Botti, P.; Villain, M.; Manganiello, S.; Gaertner, H.
Org. Lett. 2004, 6, 4861–4864; (b) Warren, J. D.; Miller, J.
S.; Keding, S. J.; Danishefsky, S. J. J. Am. Chem. Soc.
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Acknowledgements
We thank Professor R. T. Raines for helpful discussions.
We thank Dr. Teiji Chihara and his staff for elemental
analyses and Ms. Ikuko Kagawa at the Research
Resource Center of RIKEN Brain Science Institute
for the preparation of the peptides. We also thank
Ms. Akemi Takahashi for her technical assistance.
References and notes
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